Nontumorigenic Prostate Epithelial Cell Line Research Articles

Abstract A109: MicroRNA-4719 and microRNA-6756-5p correlate with aggressive prostate cancer progression in African American men through regulation of Interleukin-24

Abstract Prostate cancer (PCa) incidence and mortality are highest in African American men (AAM) and the molecular mechanisms underlying the racial disparities in PCa are unclear. Interleukin-24 (IL-24), a tumor suppressor whose expression is lost in most human cancers, correlates with cancer progression. Also, microRNAs (miRs) are dysregulated in cancers. microRNA target prediction algorithm tools identified miRs, miR-4719 and miR-6556-5p, as putative regulators of IL-24. This study elucidates the expression profile and role of miR-4719 and miR-6756-5p as regulators of IL-24 in PCa biology of AAM. Our work compares five different PCa cell lines: E006AA (AAM, indolent), E006AA-hT (AAM, aggressive), DU-145 (CM, aggressive), PC-3 (CM, aggressive) to a nontumorigenic prostate epithelial cell line, RWPE1 (CM). qRT-PCR analysis shows that miR-4719 and miR-6756-5p are significantly overexpressed in all PCa cell lines (by >2-fold) compared to RWPE-1. Both miR-4719 (>2 fold) and miR-6756-5p (>50%) are higher in aggressive PCa compared to the indolent PCa, indicating their gain could be an early event in PCa progression. Moreover, miR-4719 and miR-6756-5p are significantly overexpressed in the aggressive PCa of AAM (E006AA-hT) compared to aggressive PCas for CM (PC-3 and DU-145). Interestingly, both miR-4719 and miR-6756-5p expression were higher by (>3-fold) in the aggressive AAM cell line E006AA-hT compared to indolent AAM cell line- E006AA. Thus, miR-4719 and miR-6756-5p may play a role in racial disparity. Using oligonucleotide mimics and inhibitors, we evaluated the functional role of these miRs in the AAM cell lines, E006AA and E006AA-hT. MTT assays revealed that inhibition of miR-4719 and miR-6756-5p significantly decreases proliferation (>40%), while overexpression of both miRs increases proliferation (>20%). Inhibition of miR-4719 and miR-6756-5p further decreased the proliferation of the aggressive E006AA-hT by an extra 20%, compared to indolent E006AA. In contrast, overexpression of both miRs further increased the proliferation of E006AA-hT also by an extra 20%, compared to E006AA. Wound healing assays reveal that inhibition of miR-4719 and miR-6756-5p reduced migration by ~50% compared to the negative control (NC). Strikingly, overexpression of both miRs increased migration by at least 2- and 4-fold, respectively. We observed that IL-24 was downregulated in all PCa cells compared to RWPE-1, suggesting a possible role of these miRs in suppressing IL-24. qRT-PCR analysis show inhibition of miR-4719 and miR-6756-5p significantly increases the expression of IL-24 (by ~2-fold) in PCa cells compared to the NC. In contrast, overexpression of both miRs reduces the expression of IL-24. Our findings indicate that miR-4719 and miR-6756-5p may regulate IL-24 expression and may be biomarkers that can differentiate between indolent and aggressive PCa in AAM. Strategies to inhibit miR-4719 and miR-6756-5p expression to increase IL-24 in PCa could have therapeutic efficacy in AAM. Citation Format: Dibash K. Das, Leah Persaud, Moira Sauane. MicroRNA-4719 and microRNA-6756-5p correlate with aggressive prostate cancer progression in African American men through regulation of Interleukin-24 [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr A109.

Abstract B074: The long noncoding RNA from PVT1 exon 9 is overexpressed in prostate cancer in Black males and induces malignant transformation, invasiveness, and castration-resistance in prostate epithelial cells

Abstract Prostate cancer (PCa) is the most common non-skin cancer and the second leading cause of cancer-related death for men in the United States. PCa is the greatest source of cancer-related mortality in males of African ancestry. One of the most important susceptibility loci for cancer is the 8q24 human chromosomal region. The non-protein coding gene locus plasmacytoma variant translocation 1 (PVT1) is located at 8q24 and is dysregulated in different cancers. PVT1 gives rise to several alternatively spliced transcripts and microRNAs. There are at least twelve exons of PVT1, which make separate transcripts, and likely have different functions. The transcript from PVT1 exon 9 is significantly overexpressed in PCa tissues in comparison to normal prostate tissues obtained from Black males. Both transient and stable overexpression of PVT1 exon 9 induce significantly increased prostate epithelial cell proliferation, migration, and proliferating cell nuclear antigen (PCNA) expression. Notably, implantation into mice of a novel subline of a non-tumorigenic prostate epithelial cell line stably overexpressing PVT1 exon 9 results in the formation of malignant tumors with features characteristic of aggressive PCa. Further, PVT1 exon 9 overexpression significantly induces castration-resistance in prostate epithelial cells. Consequently, PVT1 exon 9 expression is important for PCa initiation and progression and may have potential diagnostic and therapeutic applications in PCa in Black males. Citation Format: Gargi Pal, Jeannette Huaman, Fayola Levine, Akintunde Orunmuyi, E. Oluwabunmi Olapade-Olaopa, Olorunseun O. Ogunwobi. The long noncoding RNA from PVT1 exon 9 is overexpressed in prostate cancer in Black males and induces malignant transformation, invasiveness, and castration-resistance in prostate epithelial cells [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B074.

Long Noncoding RNA from PVT1 Exon 9 Is Overexpressed in Prostate Cancer and Induces Malignant Transformation and Castration Resistance in Prostate Epithelial Cells.

Prostate cancer (PCa) is the most common non-cutaneous cancer and second leading cause of cancer-related death for men in the United States. The nonprotein coding gene locus plasmacytoma variant translocation 1 (PVT1) is located at 8q24 and is dysregulated in different cancers. PVT1 gives rise to several alternatively spliced transcripts and microRNAs. There are at least twelve exons of PVT1, which make separate transcripts, and likely have different functions. Here, we demonstrate that PVT1 exon 9 is significantly overexpressed in PCa tissues in comparison to normal prostate tissues. Both transient and stable overexpression of PVT1 exon 9 significantly induced greater prostate epithelial cell migration, as well as increased proliferation and corresponding proliferating cell nuclear antigen (PCNA) expression. Notably, implantation into mice of a non-tumorigenic prostate epithelial cell line stably overexpressing PVT1 exon 9 resulted in the formation of malignant tumors. Furthermore, PVT1 exon 9 overexpression significantly induced castration resistance. Consequently, PVT1 exon 9 expression is important for PCa initiation and progression, and holds promise as a therapeutic target in PCa.

Abstract A049: Centrosome loss and chromosomal instability in prostate tumor progression

Abstract Background: Chromosomal instability is a hallmark of cancer. Early genomic events in tumor progression include copy number variations such as tandem duplications and deletions that involve at least >100kB stretches of DNA. Although single-driver mutations are remarkably infrequent in most cancers (e.g., accounting for only 2-3% of human epithelial cancers), approximately 20% of prostate tumors are reported to display chromothripsis. Errors in mitotic chromosome segregation produce micronuclei that are susceptible to chromothripsis, and this can occur due to changes in centrosome numbers. Centrosomes are organelles that nucleate microtubule growth and, thus, determine the number of mitotic spindle poles. Normally, mitotic cells contain two centrosomes that guide formation of a bipolar spindle, ensuring that daughter cells divide within the epithelial plane and inherit an equal complement of the genome. Centrioles are the duplicating elements of centrosomes, and their loss or overduplication (known as “amplification”) promotes spindle assembly defects that lead to chromothripsis. Precise control of centriole copy number is vital in order to maintain genomic stability and normal tissue homeostasis. Methods: We developed an assay for the detection and quantitation of centrosome numbers in FFPE normal and cancer tissue and tissue culture cell lines. In addition, a 3D culture model of an immortalized nontumorigenic prostate epithelial cell line (RWPE-1) was used to characterize the progression of prostate spheroids from normal gland to high-grade prostatic intraepithelial neoplasia (HG-PIN) type structures in vitro. Genomic instability was detected by polyploidy (flow and metaphase analysis), SKY (Roswell Park Cancer Institute Cytogenetics SKY core laboratory), and micronuclei formation. Chromosome fragility was assessed by time-lapse microscopy. Results: Human prostate cancer has a readily detectable centrosome dysfunction that occurs early in the disease progression. Whereas triple-negative breast cancer specimens contained centrosome amplification as expected, surprisingly, centrosome loss was detected in early-stage prostate cancer (Gleason stage 3+3, 3+4 and 4+4) tissue specimens and in AR-positive cell lines (LnCAP, VCaP). We note that centrosome amplification was observed in AR-negative cell lines (DU145, PC3 and H660). Using TCGA data, twelve centriolar genes were either amplified, deleted, or mutated in prostate adenocarcinoma and metastatic disease. In contrast, all twelve centriolar genes were highly amplified in neuroendocrine prostate cancer. Using centrinone, a small-molecule inhibitor of Polo-like kinase 4, we blocked centriole duplication in nontumorigenic prostate cells (RWPE-1). Centrinone treatment resulted in significant genomic instability (polyploidy, copy number variation across all chromosomes, and micronuclei formation) and mitotic errors (increased mitotic index, prolonged mitosis, and increased chromosome fragility). Additionally, centrosome loss produced prostate cancer-specific phenotypes (erg overexpression, TMPRSS2 amplification, and invasive budding). Conclusions: The loss of centrosomes during early stages of prostate cancer development may account for the increased chromosomal instability manifested as polyploidy and copy number variations. Whether alterations in centrosome numbers occur in high-grade prostate cancer or metastatic lesions is unknown. Centrosomes may be promising biomarkers for advancing disease but also rational targets for personalized therapy in prostate cancer. (Supported in part by NIH grants CA 23074, CA 159406 and NIGMS R01GFM110166.) Citation Format: Mengdie Wang, Beatrice S. Knudsen, Gregory C. Rogers, Anne E. Cress. Centrosome loss and chromosomal instability in prostate tumor progression [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A049.

Proinflammatory CXCL12-CXCR4/CXCR7 Signaling Axis Drives Myc-Induced Prostate Cancer in Obese Mice.

Obesity is a prognostic risk factor in the progression of prostate cancer; however, the molecular mechanisms involved are unclear. In this study, we provide preclinical proof of concept for the role of a proinflammatory CXCL12-CXCR4/CXCR7 signaling axis in an obesity-driven mouse model of myc-induced prostate cancer. Analysis of the stromal vascular fraction from periprostatic white adipose tissue from obese HiMyc mice at 6 months of age revealed a dramatic increase in mRNAs encoding various chemokines, cytokines, growth factors, and angiogenesis mediators, with CXCL12 among the most significantly upregulated genes. Immunofluorescence staining of ventral prostate tissue from obese HiMyc mice revealed high levels of CXCL12 in the stromal compartment as well as high staining for CXCR4 and CXCR7 in the epithelial compartment of tumors. Prostate cancer cell lines derived from HiMyc tumors (HMVP2 and derivative cell lines) displayed increased protein expression of both CXCR4 and CXCR7 compared with protein lysates from a nontumorigenic prostate epithelial cell line (NMVP cells). CXCL12 treatment stimulated migration and invasion of HMVP2 cells but not NMVP cells. These effects of CXCL12 on HMVP2 cells were inhibited by the CXCR4 antagonist AMD3100 as well as knockdown of either CXCR4 or CXCR7. CXCL12 treatment also produced rapid activation of STAT3, NFκB, and MAPK signaling in HMVP2 cells, which was again attenuated by either AMD3100 or knockdown of CXCR4 or CXCR7. Collectively, these data suggest that CXCL12 secreted by stromal cells activates invasiveness of prostate cancer cells and may play a role in driving tumor progression in obesity. Targeting the CXCL12-CXCR4/CXCR7 axis could lead to novel approaches for offsetting the effects of obesity on prostate cancer progression. Cancer Res; 77(18); 5158-68. ©2017 AACR.

Abstract P6-01-05: Novel cytotoxic RNA aptamers that distinguish between metastasis-prone and indolent breast and prostate cancers

Abstract Background: Prostate and breast cancers are, respectively, the most common malignancies diagnosed in men and women worldwide. These cancers develop in different organs but have significant biological similarities: both are typically hormone-dependent, and both require early detection and treatment, as metastatic disease is incurable. At the same time, early stage tumors are often over-treated. Better markers for tumor aggressiveness would help to optimize treatment strategies in both breast and prostate cancer. Objective: Develop high affinity nucleic acid oligomers (aptamers) that can distinguish between indolent tumors that will remain organ-confined and those with heightened potential to metastasize. Methods: We performed subtractive RNA Cell-SELEX to select for surface ligands specific to aggressive tumors, using as a positive selector the highly metastasis-competent LN3 subclone of prostate cancer cell line LNCaP, and as negative selectors parental LNCaP and a non-metastasizing subclone, Pro5. . The RNA aptamer pool was PCR amplified from a 40-mer random nucleotide cDNA library with appropriate flanking sequences, and transcribed in vitro. After 11 SELEX cycles, aptamer pools from cycles 0, 4, 9, and 11 were subjected to high-throughput sequencing. Eight aptamers, representing 4 sequence families, were chosen for further study. Representative relevant and irrelevant aptamers were labeled with Cy3 and used to stain LNCaP-LN3 and LNCaP-Pro5 in culture and as xenografts in NOD-SCID-gamma mice. Additional cell and tumor lines from both breast and prostate cancer were used for validation. Results: Two aptamers bound avidly to the surface of the aggressive LNCaP-LN3 subclone, both in culture and in fixed xenograft tumors, but not to the indolent Pro5 subclone. Aptamer binding led to rapid and specific cytotoxicity in vitro but had no effect on other cell lines to which the aptamer did not bind. The same aptamers showed similar high specificity for multiple other metastasis-competent cancer cells, including the prostate adenocarcinoma PC-3 and PC-3ML subclones, breast cancer cell lines MDA-MB436 and MDA-MB231, and the primary dissociated breast tumor DT28 , while exhibiting no detectable binding to the non-metastasizing MCF-7 breast cancer cell line and DT22 primary dissociated breast tumor cells, and the non-tumorigenic prostate epithelial cell line RPWE-1. Conclusion: We identified RNA aptamers that specifically bind to metastasis-prone prostate cancer and breast cancer cell surface targets, and exert cell-specific toxicity dependent upon aptamer binding. While the target(s) remain to be identified, we propose that these aptamers may discriminate between progressive and indolent breast and prostate cancers, and may have substantial promise as anticancer agents either alone or suitably liganded to toxic moieties. Citation Format: Bishopric NH, Speransky S, Serafini P, De la Fuente AC, Bicciato S, El-Ashry D, Lippman ME. Novel cytotoxic RNA aptamers that distinguish between metastasis-prone and indolent breast and prostate cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-05.

Abstract 187: miR-1205/FRYL as a novel regulatory mechanism in androgen-independent prostate cancer

Abstract Prostate Cancer (PCa) is the second most common cause of cancer-related deaths in American men, mostly related to the development of androgen-independent PCa (AIPC). Understanding of the molecular mechanisms underlying the development of AIPC is critical to discovering more effective therapeutic strategies for AIPC. MicroRNAs (miRNAs) are short non-coding RNAs that play an important role in post-transcriptional regulation and have been implicated in cancer development. A recently annotated miRNA is miR-1205 encoded by the long non-coding PVT1 gene locus which is located at the prostate cancer susceptibility chromosomal region, 8q24. The role of miR-1205 in PCa is, however, unknown. This study aimed to determine whether miR-1205 plays an essential role in PCa progression. A panel of five prostate cell lines with a diversity of PCa characteristics was used. RWPE1, a non-tumorigenic prostate epithelial cell line, WPE1-NA22, an indolent PCa cell line derived from RWPE1 and three aggressive PCa cell lines (PC3, VCaP and MDA PCa 2b) were used. Expression of miR-1205 was determined using real-time quantitative polymerase chain reaction. We evaluated the effect of the loss or gain of miR-1205 by transfecting the oligonucleotide inhibitor or oligonucleotide mimic respectively. Effect of loss or gain of miR-1205 on proliferation and migration was measured using MTT and wound healing assays, respectively. The miSVR computer algorithm was used to ascertain putative targets. Effect of transfection of oligonucleotide mimic on mRNA expression of the putative targets was used to confirm the miR-1205 target. Our study indicates that the expression of miR-1205 is markedly less in PCa cells (WPE1-NA22, MDA PCa 2b, PC3, and VCaP) in comparison to the RWPE1 non-tumorigenic prostate epithelial cell line. The expression of miR-1205 was further significantly less in AIPC cells (PC3) compared to the other PCa cells. Loss of miR-1205 significantly increased cell proliferation in prostate epithelial cells. Exogenous miR-1205 significantly decreased cell proliferation in prostate epithelial cells. Also, loss of miR-1205 promoted migration in PCa cells by nearly 3 folds. The miSVR computer algorithm identified FRY-like (FRYL) as a putative target of miR-1205. Transfecting the oligonucleotide mimic of miR-1205 into androgen-independent PC3 cells leads to significant decrease in the expression of FRYL, suggesting that FRYL is a direct target of miR-1205. Therefore, miR-1205 regulates proliferation and migration of prostate epithelial cells, and loss of miR-1205 promotes a tumorigenic phenotype in PCa. Moreover, miR-1205 targets FRYL in AIPC. Consequently, strategies to increase miR-1205 or target FRYL may have therapeutic potential in AIPC. Citation Format: Victoria Durojaiye, Adeodat Ilboudo, Fayola Levine, Joseph Osborne, Jong Y. Park, Olorunseun O. Ogunwobi. miR-1205/FRYL as a novel regulatory mechanism in androgen-independent prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 187. doi:10.1158/1538-7445.AM2015-187

Abstract 175: miR-1207-3p-induced downregulation of fibronectin is a novel regulatory pathway in prostate cancer

Abstract Prostate cancer (PC) is the most commonly diagnosed male cancer in the western world. The molecular mechanisms underlying its development and progression are still unclear. Recent studies have established an important role for micro RNAs (miRNAs) in human cancers. miRNAs play a crucial role in post-transcriptional gene regulation. Some miRNAs have been implicated in PC. MicroRNA-1207-3p (miR-1207-3p) is encoded at the non-protein coding gene locus PVT1 on the 8q24 human chromosomal region, an established PC susceptibility locus. This study aims to determine the role of miR-1207-3p in PC. miR-1207-3p expression was investigated in a panel of five PC cell lines representative of different characteristics of PC: WEP1-NA22 (derived from RWPE1, indolent, androgen dependent, White male), VCaP (aggressive, androgen-dependent, White male), MDA PCa 2b (aggressive, androgen-dependent, Black male), and PC-3 (aggressive, androgen-independent, White male), and compared to a non-tumorigenic prostate epithelial cell line, RWPE1 (White male). To evaluate the role of miR-1207-3p, effects of gain of expression and loss of expression on cell proliferation, migration and apoptosis were performed using an oligonucleotide mimic, an oligonucleotide inhibitor or a non-targeting negative control oligonucleotide. A target prediction algorithm was used to identify a putative target of miR-1207-3p, and confirmation of the target was done by assessing the effect of miR-1207-3p mimic on putative target expression using real-time PCR (qPCR) and western blotting. miR-1207-3p is significantly underexpressed (by almost 2-fold) in both indolent (WPE1-NA22) and aggressive (MDA PCa 2b, PC-3 and VCaP) human PC cell lines in comparison to normal prostate epithelial cells (RWPE1). This indicates that loss of miR-1207-3p may be an early event in PC development. Increased expression of miR-1207-3p in PC cells significantly inhibits proliferation, induces apoptosis and inhibits migration. Inhibition of miR-1207-3p expression in prostate epithelial cells causes a significant increase in proliferation, and a significant inhibition of apoptosis. Inhibition of miR-1207-3p expression led to a 4-fold increase in the migratory capacity of prostate epithelial cells. The miSVR computer algorithm identified fibronectin as a putative molecular target of miR-1207-3p. Forced expression of miR-1207-3p in prostate epithelial cells significantly reduced fibronectin expression. Fibronectin is significantly overexpressed in the PC cell lines in comparison to the non-tumorigenic prostate epithelial cell line. miR-1207-3p may have tumor suppressive effects in PC. These effects may be via direct targeting of fibronectin. In conclusion, we uncovered a miR-1207-3p-induced downregulation of fibronectin as a possible novel regulatory pathway in PC. Therefore, strategies to increase miR-1207-3p expression in PC cells may have therapeutic efficacy in PC. Citation Format: Dibash K. Das, Adeodat Ilboudo, Joseph R. Osborne, Olorunseun O. Ogunwobi. miR-1207-3p-induced downregulation of fibronectin is a novel regulatory pathway in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 175. doi:10.1158/1538-7445.AM2015-175

Abstract LB-16: DACH1 inhibited prostate cancer cellular proliferation and interleukin-6 signaling

Abstract The Retinal Determination Gene Network (RDGN), consisting of dachshund (dac), eyes absent (eya), and sin oculis (so), is best known for its role in eye specification. Recently, coordinate abnormal expression of the DACH1/Eya/Six complex has been observed in human tumors supporting a tumor promoting role of Six and Eya family members, and a tumor-suppressor function of DACH. Oncogenes sustain host-cell interactions, promoting invasiveness, recruiting inflammatory mediators and developing angiogenic networks. Ras and Myc promote tumor angiogenesis through up-regulation of VEGF and the inflammatory mediator, interleukin-8(IL-8) and interleukin-6 (IL-6). Altered expressions of the IL-6 and IL-8 genes are seen in many cancers including prostate cancer. Hormone-resistant prostate carcinoma cell lines produce large amounts of IL-6 and IL-8 compared to hormone responsive prostate cancer cell lines. The expression of IL-8 and IL-6 are linked to poor prognosis and metastatic phenotype in prostate cancer. Recent finding demonstrated that IL-6 regulates the dynamic equilibrium between cancer stem cells and non-stem cancer cells in human breast and prostate cancer. Moreover, IL-6 alone can transform non-tumorigenic benign prostate epithelial cells and further progress to invasive phenotype. We had previously reported that the expression of DACH1 was reduced in human prostate cancer and DACH1 inhibited androgen signaling transduction. To explore the role of DACH1 in androgen resistant prostate cancer, PC3 cells stably expressing DACH1 were established. DACH1 inhibited cellular proliferation evaluated by MTT assay and growth curve. Microarray analyses demonstrated DACH1 inhibited mRNA expression of GRO, IL-6, IL-8 by 75%, 80% and 90%, respectively. However, there was no change of mRNA expression for IL-6 and IL-8 receptor. Ectopic expression of DACH1 in PC-3 cells significantly decreased tumor growth compared with vector control group (p<0.001), and DS domain was required. It has been reported that stable expression IL-6 in SV40T immortalized non-tumorigenic prostate epithelial cell line p69 induced EMT and acquired malignant phenotypes through autocrine loop IL6 and IGF-1R signaling. To further test the functional interaction of DACH1 with IL-6, a table cells expressing IL-6 alone or IL-6 with DACH1 were deployed. Ectopic expression of DACH1 attenuated activation of IGF-1R by IL-6 as evaluated by phosphorylation of IGF-1R. Subcutaneous implantation study showed an in vivo tumor growth initiated by IL-6 in p69 was inhibited by more than 60% with the expression of DACH1. Together, our studies suggest that DACH1 is a potential tumor suppressor of prostate through repression of IL-6 signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-16. doi:1538-7445.AM2012-LB-16

Recurrent deletion of CHD1 in prostate cancer with relevance to cell invasiveness

Though prostate cancer is often indolent, it is nonetheless a leading cause of cancer death. Defining the underlying molecular genetic alterations may lead to new strategies for prevention or treatment. Towards this goal, we performed array-based comparative genomic hybridization (CGH) on 86 primary prostate tumors. Among the most frequent alterations not associated with a known cancer gene, we identified focal deletions within 5q21 in 15 out of 86 (17%) cases. By high-resolution tiling array CGH, the smallest common deletion targeted just one gene, the chromatin remodeler chromodomain helicase DNA-binding protein 1 (CHD1). Expression of CHD1 was significantly reduced in tumors with deletion (P=0.03), and compared with normal prostate (P=0.04). Exon sequencing analysis also uncovered nonsynonymous mutations in 1 out of 7 (14%) cell lines (LAPC4) and in 1 out of 24 (4%) prostate tumors surveyed. RNA interference-mediated knockdown of CHD1 in two nontumorigenic prostate epithelial cell lines, OPCN2 and RWPE-1, did not alter cell growth, but promoted cell invasiveness, and in OPCN2-enhanced cell clonogenicity. Taken together, our findings suggest that CHD1 deletion may underlie cell invasiveness in a subset of prostate cancers, and indicate a possible novel role of altered chromatin remodeling in prostate tumorigenesis.

Abstract A62: Monensin-induced oxidative stress reduces prostate cancer cell motility and cancer stem cell markers.

Abstract Therapeutic options for prostate cancer are limited and treatment responses to currently existing therapies are often unsatisfactory. Thus, there is an urgent need for novel agents to target advanced and metastatic prostate cancer cells. We have recently carried out a chemical-biological high-throughput screening of 4,910 known drugs and drug-like molecules in four prostate cancer cell models and two non-tumorigenic prostate epithelial cell lines to identify prostate cancer cell growth selective inhibitors. Only four compounds, antibiotic ionophore monensin, aldehyde dehydrogenase (ALDH) inhibitor disulfiram, histone deacetylase inhibitor trichostatin A and fungicide thiram inhibited selectively cancer cell growth at nanomolar concentrations. The mechanistic studies indicated that monensin inhibited prostate cancer cell growth by inducing oxidative stress and apoptosis. In addition, monensin reduced androgen receptor signaling, showed a synergistic anti-proliferative effect with anti-androgens as well as reduced the levels of MYC and ERG oncogenes and reduced the activity of ALDH in prostate cancer cells. Moreover, antioxidant vitamin C rescued the monensin induced growth inhibition, indicating that oxidative stress plays a key role in the antineoplastic effect of monensin in cultured prostate cancer cells. Our previous Connectivity Map results indicated that monensin has agonistic effects to NF-κB inactivator and oxidative stress inducer niclosamide. Here, we show that monensin indeed reduced the activity of NF-κB pathway. NF-κB maintains cellular antioxidant defence capacity and its inhibition induces oxidative stress as well as reduces tumorigenesis, metastasis and cancer stem cell potential. Cancer stem cells have a controlled redox balance system including high ALDH and CD44 expression which protect cancer stem cells from oxidative stress. Our results confirmed that monensin reduced the cancer stem cell markers in prostate cancer cells. Moreover, monensin induced epithelial cell differentiation shown as well as reduced motility in cultured prostate cancer cells, suggesting that monensin inhibits prostate tumorigenesis by multiple ways. Furthermore, the steroid profiling indicated that monensin increases the levels of oxidative stress inducing steroids and reduces androgen precursors in cultured prostate cancer cells. In conclusion, our results suggest that impairing the redox control, which has a crucial role in cancer cells enabling survival under high intracellular ROS, is a potent way to target prostate cancer cells and potentially also prostate cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A62.

Abstract A21: DACH1 blocks prostate cancer cell growth and interleukin-6 signaling

Abstract In Drosophila, eye development is coordinately regulated by the retinal determination gene network (RDGN), mainly consisting of dachshund (dac), eyes absent (eya), and sin oculis (so). The mammalian homologues are Dach, Eya, and Six, respectively. RDGN, although best known for its role in eye specification, is essential for the development of many organs including retinal, kidney, brain, and limb in both flies and mammals. Recently, coordinate abnormal expression of the DACH1/Eya/Six complex has been observed in human tumors supporting a tumor promoting role of Six and Eya family members, and a tumor-suppressor function of DACH. Oncogenes sustain host-cell interactions, promoting invasiveness, recruiting inflammatory mediators and developing angiogenic networks. Ras and Myc promote tumor angiogenesis through up-regulation of VEGF and the inflammatory mediator, interleukin-8(IL-8) and interleukin-6 (IL-6). Our previous studies demonstrate DACH1 inhibits migration of MCF10A cells transformed with distinct oncogenes (Ha-Ras, c-Raf, c-Myc, and ErbB2) as well as the highly metastatic MDA-MB-231 human breast cancer cell line. An unbiased proteomic approach to identify candidate proteins regulated by DACH1 expression, using antibody array analysis, demonstrated DACH1 expression correlated with reduced secretion of IL-8 and the related chemokines IL-1, IL-6, GRO, and MIP1 (α, β, γ) from Ras or c-Myc transformed MCF-10A cells and repressed IL-8 promoter activity. Altered expressions of the IL-6 and IL-8 genes are seen in many cancers including prostate cancer. Hormone-resistant prostate carcinoma cell lines produce large amounts of IL-6 and IL-8 compared to hormone responsive prostate cancer cell lines. The expression of IL-8 and IL-6 are linked to poor prognosis and metastatic phenotype in prostate cancer. Recent finding demonstrated that IL-6 regulates the dynamic equilibrium between cancer stem cells and non-stem cancer cells in human breast and prostate cancer. IL-6 alone can transform non-tumorigenic benign prostate epithelial cells and further progress to invasive phenotype. To explore the role of DACH1 in androgen resistant prostate cancer, PC3 cells stably expressing DACH1 were established. DACH1 inhibited proliferation evaluated by MTT assay and growth curve. Microarray analyses demonstrated DACH1 inhibited mRNA expression of GRO, IL-6, IL-8 by 75%, 80% and 90%, respectively. However, there was no change of mRNA expression for IL-6 and IL-8 receptor. It has been reported that stable expression IL-6 in SV40T immortalized non-tumorigenic prostate epithelial cell line p69 induced EMT and acquired malignant phenotypes through autocrine loop IL6 and IGF-1R signaling. To further test the functional interaction of DACH1 with IL-6, a table cells expressing IL-6 alone or IL-6 with DACH1 were deployed. Ectopic expression of DACH1 attenuated activation of IGF-1 R by IL-6 as evaluated by phosphorylation of IGF-1R. Subcutaneous implantation study showed a 60% inhibition of in vivo tumor growth by DACH1. Together, our studies suggest that loss expression of DACH1 is an important cause of prostate tumorigenesis and restoration of DACH1 expression provides a potential therapeutic application. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A21.

Abstract 1267: Metabolic sensor O-GlcNAc Transferase is a novel regulator of prostate cancer invasion and angiogenesis via regulation of FoxM1

Abstract Cancer cells universally increase glucose and glutamine consumption, leading to the altered metabolic state known as the Warburg effect. One metabolic pathway highly dependent on glucose and glutamine is the Hexosamine Biosynthetic Pathway (HBP). Increased flux through the HBP leads to increases in the post-translational addition of O-linked-β-N-acetylglucosamine (O-GlcNAc) on a diverse population of nuclear and cytosolic proteins. A number of these target proteins are implicated in cancer, yet the role of O-GlcNAc modifications in cancer remains poorly defined. Here, we show that OGT is overexpressed in prostate cancer tissue compared to normal prostate epithelium and, that OGT protein and O-GlcNAc levels are elevated in prostate carcinoma cell lines compared to non-transformed prostate cells. Decreasing O-GlcNAc levels using OGT-targeted RNAi in the metastatic prostate cancer cell line PC3-ML inhibited growth in three-dimensional (3D) culture, as well as decreased colony formation in soft agar assays. Furthermore, decreasing O-GlcNAc levels was also associated with cell cycle arrest at G1 and increased expression of the CDK inhibitors p27 and p21. However, reducing O-GlcNAc levels in the non-tumorigenic prostate epithelial cell line RWPE-1 had minimal effects on growth in 3D culture. In addition to growth inhibition, reducing O-GlcNAcation in PC3-ML cells was associated with reduced expression of matrix metalloproteinase-2 (MMP-2), MMP-9 and vascular endothelial growth factor (VEGF), resulting in the inhibition of invasion and angiogenesis. OGT regulation of invasion and angiogenesis was dependent upon regulation of the oncogenic transcription factor FoxM1, as reducing OGT expression led to increased FoxM1 proteasomal degradation. Moreover, overexpression of a FoxM1 degradation-resistant mutant abrogated OGT RNAi inhibition of invasion, MMP levels, as well as angiogenesis and VEGF expression. Finally, the use of novel second-generation small molecule inhibitor of OGT led to the similar reduction in growth, invasion and inhibition of MMPs and VEGF levels. Current experiments are underway to test whether targeting OGT blocks prostate cancer metastasis. Altogether, these data suggest that as prostate cancer cells alter glucose and glutamine levels, O-GlcNAcation serves as nutrient sensitive modification contributing to oncogenesis and suggest that OGT is positioned as a novel target for therapeutic intervention for the treatment of human prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1267. doi:10.1158/1538-7445.AM2011-1267

HOXC8 Inhibits Androgen Receptor Signaling in Human Prostate Cancer Cells by Inhibiting SRC-3 Recruitment to Direct Androgen Target Genes

HOX (homeobox) genes encode homeodomain-containing transcription factors critical to development, differentiation, and homeostasis. Their dysregulation has been implicated in a variety of cancers. Previously, we showed that a subset of genes of the HOXC cluster is upregulated in primary prostate tumors, lymph node metastases, and malignant prostate cell lines. In the present study, we show that HOXC8 inhibits androgen receptor (AR)-mediated gene induction in LNCaP prostate cancer cells and HPr-1 AR, a nontumorigenic prostate epithelial cell line. Mechanistically, HOXC8 blocks the AR-dependent recruitment of the steroid receptor coactivators steroid receptor coactivator-3 (SRC-3), and CREB binding protein to the androgen-regulated prostate-specific antigen gene enhancer and inhibits histone acetylation of androgen-regulated genes. Inhibition of androgen induction by HOXC8 is reversed upon expression of SRC-3, a member of the SRC/p160 steroid receptor cofactor family. Coimmunoprecipitation studies show that HOXC8 expression inhibits the hormone-dependent interaction of AR and SRC-3. Finally, HOXC8 expression increases invasion in HPr-1 AR nontumorigenic cells. These data suggest a complex role for HOXC8 in prostate cancer, promoting invasiveness while inhibiting AR-mediated gene induction at androgen response element-regulated genes associated with differentiated function of the prostate. A greater understanding of HOXC8 actions in the prostate and its interactions with androgen signaling pathways may elucidate mechanisms driving the onset and progression of prostate cancer.

1,25 dihydroxyvitamin D-mediated orchestration of anticancer, transcript-level effects in the immortalized, non-transformed prostate epithelial cell line, RWPE1

BackgroundProstate cancer is the second leading cause of cancer mortality among US men. Epidemiological evidence suggests that high vitamin D status protects men from prostate cancer and the active form of vitamin D, 1α,25 dihydroxyvitamin D3 (1,25(OH)2D) has anti-cancer effects in cultured prostate cells. Still, the molecular mechanisms and the gene targets for vitamin D-mediated prostate cancer prevention are unknown.ResultsWe examined the effect of 1,25(OH)2D (+/- 100 nM, 6, 24, 48 h) on the transcript profile of proliferating RWPE1 cells, an immortalized, non-tumorigenic prostate epithelial cell line that is growth arrested by 1,25(OH)2D (Affymetrix U133 Plus 2.0, n = 4/treatment per time and dose). Our analysis revealed many transcript level changes at a 5% false detection rate: 6 h, 1571 (61% up), 24 h, 1816 (60% up), 48 h, 3566 (38% up). 288 transcripts were regulated similarly at all time points (182 up, 80 down) and many of the promoters for these transcripts contained putative vitamin D response elements. Functional analysis by pathway or Gene Set Analysis revealed early suppression of WNT, Notch, NF-kB, and IGF1 signaling. Transcripts related to inflammation were suppressed at 6 h (e.g. IL-1 pathway) and suppression of proinflammatory pathways continued at later time points (e.g. IL-17 and IL-6 pathways). There was also evidence for induction of anti-angiogenic pathways and induction of transcripts for protection from oxidative stress or maintenance of cell redox homeostasis at 6 h.ConclusionsOur data reveal of large number of potential new, direct vitamin D target genes relevant to prostate cancer prevention. In addition, our data suggests that rather than having a single strong regulatory effect, vitamin D orchestrates a pattern of changes within prostate epithelial cells that limit or slow carcinogenesis.

Epigallocatechin-3-gallate (EGCG) inhibits PC-3 prostate cancer cell proliferation via MEK-independent ERK1/2 activation

Epigallocatechin-3-gallate (EGCG), a tea polyphenol, inhibits the proliferation of many cancer cell lines; however, the antiproliferative mechanism(s) are not well-characterized. The objective of this study is to identify the cellular signaling mechanism(s) responsible for the antiproliferative effects of EGCG in the PC-3 prostate cancer cell line. EGCG inhibited PC-3 cell proliferation in a concentration-dependent manner with an IC 50 value of 39.0 μM, but had no effect on the proliferation of a nontumorigenic prostate epithelial cell line (RWPE-1). Treatment of PC-3 cells with EGCG (0–50 μM) resulted in time and concentration-dependent activation of the extracellular signal-regulated kinase (ERK1/2) pathway. EGCG treatment did not induce ERK1/2 activity in RWPE-1 cells. The activation of ERK1/2 by EGCG was not inhibited using PD98059, a potent inhibitor of mitogen-activated protein kinase kinase (MEK), the immediate upstream kinase responsible for ERK1/2 activation; suggesting a MEK-independent signaling mechanism. Pretreatment of PC-3 cells with a phosphoinositide-3 kinase (PI3K) inhibitor partially reduced both EGCG-induced ERK1/2 activation and the antiproliferative effects of this polyphenol. These results suggest that ERK1/2 activation via a MEK-independent, PI3-K-dependent signaling pathway is partially responsible for the antiproliferative effects of EGCG in PC-3 cells.

Glycitein induces cellular differentiation in nontumorigenic prostate epithelial cells

that increased consumption of soy is associated with a reduced risk for prostate cancer. Soy isoflavones are thought to be responsible, in part, for this anticancer activity. Soy isoflavones have been shown to induce cellular differentiation in a number of tissues. However, isoflavone-induced differentiation in the prostate has not been examined. The present study examined the effects of the soy isoflavone, glycitein, on luminal and basal cell differentiation in a nontumorigenic prostate epithelial cell line (RWPE-1). Differentiation was characterized by inhibition of cellular proliferation, cell cycle arrest, and cytokeratin expression. Cytokeratins are differentially expressed among epithelial cell types with luminal prostate epithelial cells expressing cytokeratins 8/18 while basal epithelial cells express cytokeratins 5/14. Treatment of RWPE-1 cells with the soy isoflavones genistein, daidzein, equol, and glycitein (0-50μM) significantly inhibited cellular proliferation at 50μM and glycitein inhibited cellular proliferation at 5 and 50μM. Expression cytokeratin 18 was increased upon treatment of RWPE-1 cells with with N-(4hydroxyphenyl) retinamide (4-HPR, 1μM), while glycitein treatment (50μM, 8 d) significantly reduced expression of this luminal marker. These data suggest that glycitein may induce basal cell differentiation in the RWPE-1 cell line. Maintaining the basal cell population within the prostate may represent a novel mechanism by which soy isoflavones reduce prostate cancer risk. ABSTRACT Elizabeth A. Clubbs and Joshua A. Bomser OSU Interdisciplinary PhD program in Nutrition, The Ohio State University, Columbus OH 43210, USA

Glycitein activates extracellular signal-regulated kinase via vascular endothelial growth factor receptor signaling in nontumorigenic (RWPE-1) prostate epithelial cells

Increased consumption of soy is associated with a decreased risk for prostate cancer; however, the specific cellular mechanisms responsible for this anticancer activity are unknown. Dietary modulation of signaling cascades controlling cellular growth, proliferation and differentiation has emerged as a potential chemopreventive mechanism. The present study examined the effects of four soy isoflavones (genistein, daidzein, glycitein and equol) on extracellularsignal-regulated kinase (ERK1/2) activity in a nontumorigenic prostate epithelial cell line (RWPE-1). All four isoflavones (10 μmol/L) significantly increased ERK1/2 activity in RWPE-1 cells, as determined by immunoblotting. Isoflavone-induced ERK1/2 activation was rapid and sustained for approximately 2 h posttreatment. Glycitein, the most potent activator of ERK1/2, decreased RWPE-1 cell proliferation by 40% ( P<.01). Glycitein-induced ERK1/2 activation was dependent, in part, on tyrosine kinase activity associated with vascular endothelial growth factor receptor (VEGFR). The presence of both VEGFR1 and VEGFR2 in the RWPE-1 cell line was confirmed by immunocytochemistry. Treatment of RWPE-1 cells with VEGF 165 resulted in transient ERK1/2 activation and increased cellular proliferation. The ability of isoflavones to modulate ERK1/2 signaling cascade via VEGFR signaling in the prostate may be responsible, in part, for the anticancer activity of soy.

Isoflavone‐induced activation of extracellular signal‐regulated kinase (ERK 1/2) in non‐tumorigenic prostate epithelial cells

Epidemiological and experimental evidence suggests that increased consumption of soy is associated with a reduced risk for prostate cancer. Soy isoflavones are thought to be responsible, in part, for this anticancer activity. The present study evaluated the ability of four isoflavones (genistein, daidzein, glycitein, and equol) to modulate ERK1/2 activity in a non-tumorigenic prostate epithelial cell line (RWPE-1). ERK1/2 activity is associated with cellular proliferation and differentiation; however, the specific role of this pathway in prostate carcinogenesis is unknown. Treatment of cells with genistein, daidzein, equol, and glycitein (10μM) increased ERK1/2 activity 7.2, 7.3, 12.3, and 47-fold respectively (p<0.05), as measured by immunoblot. Isoflavone-induced ERK1/2 activation was both time and concentration dependent. Further characterization of glycitein-induced ERK1/2 activation suggests involvement of vascular endothelial and epithelial growth factor receptor tyrosine kinase activity. Interestingly, isoflavone-induced ERK1/2 activation was not observed in the highly malignant PC-3 prostate epithelial cell line. These data suggest that glycitein, despite its relatively low abundance, is a potent activator of the ERK1/2 signaling cascade in prostate epithelial cells. Funded by USDA IFAFS.